Thyrotropin (TSH)-induced production of vascular endothelial growth factor in thyroid cancer cells in vitro: evaluation of TSH signal transduction and of angiogenesis-stimulating growth factors

Thyroid tumor growth requires angiogenesis, and vascular endothelial growth factor (VEGF) has been shown to be the most important endothelial mitogen. TSH is the major thyrotropic hormone, but its impact to modulate VEGF production has not yet been studied. Several other growth factors have also been shown to affect thyroid cancer cell growth and function in vitro. Therefore, the aim of the current study was to 1) establish the effect of TSH on VEGF as well as 2) evaluate the TSH signal transduction of this effect, and 3) screen other growth factors for the ability to modulate VEGF in thyroid cancer cell lines. HTC, a follicular cancer cell line lacking endogenous TSH receptor (TSHr), its receptor positive variant (HTC TSHr), and a cell line of Huerthle cell origin (XTC) were used. After stimulation with growth factors in vitro [TSH; epidermal growth factor (EGF), IGF, placenta growth factor, TGF-alpha, TGF-beta1, fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor] cells were analyzed for VEGF gene expression by Northern blotting and for VEGF protein by enzyme immunoassay. TSHr signal transduction was evaluated by analyzing the effect of stimulators (cholera toxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate) and inhibitors (2',5'-dideoxyadenosine and staurosporine) on VEGF protein levels under basal and TSH-stimulated conditions. TSH increased VEGF mRNA and protein in a dose-dependent manner in HTC TSHr and XTC cells by up to 40%. The effects of TSH were mediated by protein kinase C (PKC), rather than protein kinase A (PKA), stimulation, because inhibition of PKC by staurosporine resulted in a decrease in VEGF production of up to 65%, whereas inhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine) resulted in only a minor decrease. TSH was not the most powerful stimulator of VEGF production. TGF-beta1 and EGF were 1.5- to 2-fold more potent. Placenta growth factor and TGF-alpha did not induce VEGF production in TSHr-positive HTC cells, whereas they did induce VEGF production in TSHr-negative HTC cells. In thyroid cancer cell lines, TSH induces VEGF production involving the PKC, rather than the PKA, pathway. However, EGF and TGF-beta increase the capacity of thyroid cancer cells to provide VEGF more effectively than TSH. In the absence of a functioning TSHr, additional growth factors, such as TGF-alpha, increase capacity for VEGF stimulation.     

Regulation of thymosin beta10 expression by TSH and other mitogenic signals in the thyroid gland and in cultured thyrocytes.

OBJECTIVE: To investigate the expression of thymosin beta10 - a small conserved acidic protein involved in the inhibition of actin polymerization - in human and experimental thyroid goiters as well as the regulation exerted by TSH on thymosin beta10 expression in thyroid follicular cells both in vivo and in vitro. DESIGN: To this aim, we have used 5 bioptic specimens from patients affected by thyroid goiter, a well known experimental model of thyroid goitrogenesis (rat fed with the drug propylthiouracil) and a cultured rat thyroid cell line (PC Cl 3 cells) as a model system. RESULTS: We report that the mRNA expression of thymosin beta10 is markedly enhanced in human goiters compared with normal thyroid. In vivo results showed that the steady-state level of thymosin beta10 mRNA is up-regulated in the thyroid gland of propylthiouracil-fed rats in parallel with follicular cell proliferation: iodide administration to goitrous rats, which induced a marked involution of thyroid hyperplasia, reduced the mRNA level of thymosin beta10. Finally, in vitro studies showed that in cultured rat thyrocytes, the expression of thymosin beta10 mRNA is induced in a time- and dose-dependent manner by the activation of pathways which are mitogenic for thyroid cells (i.e. the protein kinase (PK) A and PKC pathways). CONCLUSION: Taken together, the findings reported here demonstrate that thymosin beta10 expression is regulated by extracellular signals that stimulate growth of thyroid cells both in vitro and in vivo, and suggest a role for this protein in thyroid diseases characterized by proliferation of follicular cells.     

Thyrotropin-dependent proliferation of in vitro rat thyroid cell systems

This review is focused on the most recent knowledge on growth control of rat thyroid cell lines. We analyzed the effect of mitogenic as well as inhibitory agents, but mainly the proliferative effect elicited by thyrotropin (TSH). The classic cAMP-dependent protein kinase (PKA) signal transduction pathway involved in TSH-mediated cell growth is analyzed exhaustively. We have also reviewed new concepts about the participation of other effectors such as small GTPases and phosphatidyl inositol-3-kinase (PI3-K) and the new data about the existence of a cAMP-dependent but PKA-independent pathway. Finally, we give information about TSH induction of cell cycle-related genes, such as G1 cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors.     

Tonic effect of endogenous TSH on the in vitro thyroid cAMP response to TSH

The present study was undertaken to compare the effects of 3,5,3'-triiodothyronine (T3) alone and T3 plus bovine thyrotrophin (bTSH) given chronically in vivo on the TSH-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in a mouse thyroid in vitro. Mice were given T3 (5 micrograms/ml) in drinking water for 4 days. The thyroid cAMP concentrations after an incubation with 10 mU/ml of TSH for 10 min were decreased by 50% in T3-treated mice as compared to the control. In the second experiment, mice were given T3 alone or T3 plus 0.5 mU of bTSH ip daily for 4 days. The combined treatment with T3 and TSH partially restored the reduction of cAMP response to TSH that was induced by T3 alone. In the third experiment, mice were given T3 alone for 7 days, or T3 for 7 days plus TSH for the last 3 days. The reduced cAMP response to TSH induced by T3 alone was again partially restored by the concomitant treatment with TSH. These results indicate 1) that the capacity of the thyroid cAMP to respond to TSH is regulated, at least in part, by a trophic effect of endogenous TSH and 2) that the impaired capacity caused by a loss of tonic effect of endogenous TSH is reversible.     

Effects of TSH on iodide transport by mouse thyroid lobes in vitro.

Thyroidal iodide concentration by in vitro mouse thyroid lobes was studied by determining the equilibrium tissue/medium iodide, concentration ratio (T/M[I-]), THYROIDAL I- influx, and thyroidal I-efflux in the presence and absence of ClO4-. Chronic thyroid stimulation by low dietary iodine increased the T/M[I-], and the increase was linearly related to I- influx. There was no difference in efflux rate when animals fed low and high iodine diets were compared, although the efflux with C104- added to block influx was increased by low iodine diet. Addition of TSH in vitro caused a delayed fall in T/M[I-] with a similar TSH concentration-dependence as colloid droplet formation. The TSH effect was mimicked by exogenous cyclic AMP but could be dissociated from stimulation of hormone release by colchicine. Thyroidal I- efflux was increased up to 20-fold by C104-. In the presence of C104- short-term and equilibrium-labeled I- exited at the same rate, but in the absence of C104- short-term-labeled I- efflux was consistently higher. The TSH-induced fall in T/M I- could be accounted for by increased iodide efflux. As TSH also increased efflux when influx was blocked by C104-, the TSH effect would seem due to an increased intrinsic follicular leakiness.     

Regulation of cell proliferation and malignant potential by irisin in endometrial,colon, thyroid and esophageal cancer cell lines

Objective

Irisin is a novel hormone that has been proposed to mediate the beneficial effects of exercise on metabolism, including body weight regulation and insulin resistance. No previous studies have evaluated whether irisin may regulate cell proliferation and malignant potential of obesity-related cancer cell lines.

Materials/Methods

Cell proliferation and malignant potential i.e. cell adhesion and colony formation were studied in vitro using human and mouse obesity-related cancer cell lines i.e. endometrial (KLE and RL95-2), colon (HT29 and MCA38), thyroid (SW579 and BHP7) and esophageal (OE13 and OE33).

Results

We observed that, in contrast to metformin, cell proliferation is not regulated by irisin in a dose-dependent manner in human and mouse obesity-related cancer cell lines. Specifically, physiological (5 to 10 nmol/L) and high physiological/pharmacological (50 to 100 nmol/L) concentrations of irisin had no effect on cell proliferation when compared to control in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines. Also, we observed that, in contrast to metformin, neither physiological nor high physiological/pharmacological concentrations of irisin regulate cell adhesion and/or colony formation in human and mouse endometrial, colon, thyroid and esophageal cancer cell lines.

Conclusions

Our data suggest that irisin, in physiological and high physiological/pharmacological concentrations, has no in vitro effect on cell proliferation and malignant potential of obesity-related cancer cell lines. Future work is needed to determine the regulation of irisin levels and any physiological effects it may have on obesity-related cancers in vivo in animals and humans.     

Regulation of smooth muscle proliferation by heparin in vitro and in vivo

Smooth muscle cell proliferation is central to the development of atherosclerotic plaques, intimal thickening, and recurrent stenosis in arteries following surgical reconstruction. The factors that might limit this process remain poorly defined. Studies reported recently from several laboratories suggest that heparin administered in pharmacological doses can suppress proliferation of smooth muscle cells. Furthermore, heparin-like molecules are synthesized by vascular wall cells, inhibit smooth muscle growth in vitro, and might act to regulate smooth muscle growth within the arterial wall.     

Estrogen action in vitro: regulation of thyroid stimulating and other pituitary hormones in cell cultures.

Primary cell cultures of ovine pituitaries can maintain production of thyroid stimulating hormone (TSH) for as long as 24 days. These cultures responded in a normal fashion to thyroxine by decreasing TSH secretion. Addition of thyrotropin releasing hormone increased TSH secretion. Physiologic levels of estradiol-17 beta (10(-11)-10(-9)M) produced a five-fold increase in secretion of TSH and a two-fold increase in intracellular TSH concentration in cell cultures. Common estrogens, but not common progestins, androgens and glucocorticoids affected TSH production. Markedly different effects of estrogen in the pituitary on follicle stimulating, luteinizing and thyroid stimulating hormones and prolactin are discussed in terms of current models of estrogen action.     

TSH, theophylline and cyclic AMP: in vitro thyroid activity in aging rats.

In vitro thyroid accumulation of cyclic 3',5'-adenosine monophosphate (cyclic AMP) and release of triiodothyronine (T₃) and thyroxine (T₄) in response to TSH, theophylline or cyclic AMP treatment was assessed in 60- and 340-day-old male rats. Plasma levels of T₃ and T₄ at the time of sacrifice were determined. Mature animals exhibited significantly lower plasma T₃ and T₄ levels but slightly elevated in vitro secretory rates of T₃ and T₄. TSH stimulation elicited little effect in the mature gland in terms of cyclic AMP accumulation or thyroid hormone release. Conversely, cyclic AMP enhanced in vitro thyroid hormone release in both age groups. The data suggest an age-related alteration in thyroid responsiveness to TSH which in turn may be a function of changes in the thyroidal adenylate cyclase-cyclic AMP-phosphodiesterase system. Evidence is also presented which suggests either cyclic AMP-mediated T₄ to T₃ conversion or a differential action of the nucleotide upon T₃ as compared to T₄ synthesis.     

Immeasurably low and non-TRH-stimulatable TSH associated with normal I-123 uptake in two goitrous euthyroid patients: possible existence of other thyroid-hormone regulated thyroid stimulators other than TSH

We described two euthyroid patients with normally functioning goiters, but with persistently undetectable and non-stimulatable TSH levels. Subject 1 was a 64-year-old woman with a large diffuse goiter who has been clinically and biochemically euthyroid without any medication for at least 19 years. Subject 2 was a 31-year-old woman with a small diffuse goiter who has been euthyroid for 4 years. Both patients had persistently undetectable levels of serum TSH, TSH receptor antibodies (TRAb) and thyroid stimulating antibodies (TSAb). Their basal TSH levels were very low and their T3 responses to TRH were very diminished or absent. In contrast, the basal levels of the other pituitary hormones and their responses to LHRH, GRH and CRH stimulation were all within normal limits in both patients. MRI images of pituitary glands, 123I thyroid uptake, and thyroid scans were normal. Ectopic thyroids were not detected on (99m)TcO4- and 123I total body scans. Factors interfering with the measurement of TSH were excluded by recovery studies. In subject 1 a T3-suppression test was positive and a perchlorate discharge test was negative. In subject 2 a T3-suppression test was negative. Euthyroid Graves' disease, subclinical hyperthyroidism, destructive thyroiditis, thyrotoxicosis of extrathyroid origin, central hypothyroidism, and nonthyroidal illness were all ruled out by these observations. These results suggest that an unknown factor, such as thyrostimulin, but not TSH or TSAb, stimulates the thyroid and maintains euthyroidism, and may have a role in the regulation of the hypothalamus-pituitary-thyroid axis.     

Induction of pancreatic acinar cell proliferation by thyroid hormone

Thyroid hormone is known to elicit diverse cellular and metabolic effects in various organs, including mitogenesis in the rat liver. In the present study, experiments were carried out to determine whether thyroid hormone is able to stimulate cell proliferation in another quiescent organ such as the pancreas. 3,5,3'-L-tri-iodothyronine (T3) added to the diet at a concentration of 4 mg/kg caused a striking increase in nuclear bromodeoxyuridine (BrdU) incorporation of rat acinar cells 7 days after treatment (the labeling index was 46.7% in T3-treated rats vs 7.1% in controls). BrdU incorporation was limited to the acinar cells, with duct cells and islet cells being essentially negative. The increase in DNA synthesis was accompanied by the presence of several mitotic figures. Histological examination of the pancreas did not exhibit any sign of T3-induced toxicity. Determination of the apoptotic index, measurement of the serum levels of alpha-amylase and lipase, and glycemia determination did not show any increase over control values, suggesting that the enhanced proliferation of acinar cells was a direct effect induced by T3 and not a regenerative response consequent to acinar or beta-cell injury. Additional experiments showed that DNA synthesis was induced as early as 2 days after T3 treatment (the labeling index was 9.4 vs 1.9% in controls) and was associated with increased protein levels of cyclin D1, cyclin A and proliferating cell nuclear antigen, with no substantial differences in the expression of the cyclin-dependent kinase inhibitor p27. The mitogenic effect of T3 on the pancreas was not limited to the rat, since extensive acinar cell proliferation was also observed in the pancreas of mice treated with T3 for 1 week (the labeling index was 28% in T3-treated mice vs 1.8% in controls). Treatment with three other ligands of nuclear receptors, ciprofibrate, all-trans retinoic acid and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, induced little or no pancreatic cell proliferation. These results demonstrated that T3 is a powerful inducer of cell proliferation in the pancreas and suggested that pancreatic acinar cell proliferation by selected agents may have potential for therapeutic use.     

Morphometry and cell proliferation in endoscopic biopsies: evaluation of a technique

A simple method for the quantification of intestinal epithelial cell proliferation in humans was evaluated. Endoscopic biopsy specimens were stained and microdissected, and the number of mitoses per gland or crypt was determined, as was the area of these units. The technique could readily be applied to tissue from the stomach, small intestine colon, and rectum. The number of mitoses and the size of the proliferative compartments increased caudally from the stomach to the cecum in humans. There was a good correlation between area and mitoses per gland or per crypt (r = 0.89; P less than 0.001), confirming the general equivalence between proliferative rate and compartment size. The method was validated by comparing microdissection-derived data with data previously obtained as part of an autoradiography-based study in the dog. This showed that similar differences in proliferation and crypt cell mass could be obtained but in less than one sixth of the time taken to score autoradiographs. It is concluded that this method for the estimation of gastrointestinal epithelial proliferation correlates well with other well-established techniques, confers speed as well as accuracy, has an all encompassing denominator, and can thus avoid many of the problems associated with the quantification of tissue sections.     

Regulation of epidermal growth factor receptors by TSH, and effects of EGF, TSH and phorbol ester on DNA synthesis in cultured porcine thyroid cells

Using porcine thyroid cells of primary monolayer culture, this study was conducted to clarify the nature and characteristics of epidermal growth factor (EGF) receptors on porcine thyroid cells and also to investigate the effects of EGF, TSH and phorbol ester on DNA synthesis. Receptors for EGF on porcine thyroid cells exist in two forms which differ in both affinity and capacity. Scatchard analysis of saturation binding assay performed at 4 degrees C for 6h indicates that there is a high affinity class of receptors with low capacity (K1 = 4.70 +/- 0.90 X 10(-9)M and 8,600 +/- 1,200 sites/cell) and low affinity receptors with high capacity (K2 = 2.24 +/- 1.17 X 10(-7)M and 65,500 +/- 18,000 sites/cell) on the cells cultured for 4 days in the absence of TSH. When thyroid cells were cultured in the presence of various concentrations of TSH (0 approximately 50 mU/ml) and for various times (0 approximately 96 h) with TSH (10 mU/ml), specific EGF binding to the cells increased dose- and time-dependently. On TSH (10 mU/ml)-treated cells for 4 days, two kinds of EGF receptors, i.e. high affinity and low capacity (K1 = 5.39 +/- 1.75 X 10(-9) M and 17,200 +/- 2,500 sites/cell) and low affinity and high capacity (K2 = 1.70 +/- 1.40 X 10(-7)M and 76,300 +/- 17,900 sites/cells), were resolved. The results indicate that TSH can modulate EGF receptors by increasing the number of high affinity sites on porcine thyroid cells. Next, using [Me-3H] thymidine incorporation into TCA precipitable materials for 48h, we studied the biological effects of EGF, TSH and phorbol myristate acetate (one of the potent phorbol esters) on DNA synthesis. Both EGF and PMA can promote [Me-3H] thymidine incorporation. Maximal responses were obtained with EGF ranging from 10(-9) to 10(-7)M and with PMA from 10(-9) to 10(-7)M. In contrast, TSH inhibits [Me-3H] thymidine incorporation dose-dependently. Almost the same results were obtained by these agents on TSH (10 mU/ml)-treated cells for 4 days, but EGF stimulated cell growth at a lower concentration of 10(-11)M, which was possibly related to an increase of high affinity receptor numbers. The growth promoting effect of EGF and PMA in combination was additive, and TSH suppressed the cell growth in the concomitant presence of EGF and PMA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cerivastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme a reductase, inhibits endothelial cell proliferation induced by angiogenic factors in vitro and angiogenesis in in vivo models

Cerivastatin is an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. It inhibits the biosynthesis of cholesterol and its precursors: farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGPP), which are involved in Ras and RhoA cell signaling, respectively. Statins induce greater protection against vascular risk than that expected by cholesterol reduction. Therefore, cerivastatin could protect plaque against rupture, an important cause of ischemic events. In this study, the effect of cerivastatin was tested on angiogenesis because it participates in plaque progression and plaque destabilization. Cerivastatin inhibits in vitro the microvascular endothelial cell proliferation induced by growth factors, whereas it has no effect on unstimulated cells. This growth arrest occurs at the G(1)/S phase and is related to the increase of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). These effects are reversed by GGPP, suggesting that the inhibitory effect of cerivastatin is related to RhoA inactivation. This mechanism was confirmed by RhoA delocalization from cell membrane to cytoplasm and actin fiber depolymerization, which are also prevented by GGPP. It was also shown that RhoA-dependent inhibition of cell proliferation is mediated by the inhibition of focal adhesion kinase and Akt activations. Moreover, cerivastatin inhibits in vivo angiogenesis in matrigel and chick chorioallantoic membrane models. These results demonstrate the antiangiogenic activity of statins and suggest that it may contribute to their therapeutic benefits in the progression and acute manifestations of atherosclerosis.     

Regulation of hypothalamic expression of KiSS-1 and GPR54 genes by metabolic factors: analyses using mouse models and a cell line

It is well established that reproductive function is metabolically gated. However, the mechanisms whereby energy stores and metabolic cues influence fertility are yet to be completely deciphered. Recently, the hypothalamic KiSS-1/GPR54 system has emerged as a fundamental regulator of the gonadotropic axis, which conveys the modulatory actions of sex steroids to GnRH neurons. Evidence is also mounting that KiSS-1 neurons may also represent the link between systemic metabolic signals and central control of reproduction. To further explore this possibility, we examined the impact of changes in energy status and key metabolic regulators on the hypothalamic expression of KiSS-1 and GPR54 genes, using different mouse models and the hypothalamic cell line N6. Time-course analysis of the effects of short-term fasting revealed a rapid (12- and 24-h) decline in KiSS-1 and GPR54 mRNA levels, which preceded that of GnRH (48 h). In contrast, diet-induced obesity or obesity associated with leptin deficiency (ob/ob vs. wild-type mice) failed to induce overt changes in hypothalamic expression of KiSS-1 and GPR54 genes. However, leptin infusion of ob/ob mice evoked a significant increase in KiSS-1 and GPR54 mRNA levels compared with pair-fed controls. Moreover, leptin, but not insulin or IGF-I, stimulated KiSS-1 mRNA expression in the mouse hypothalamic cell line N6. In addition, neuropeptide Y (NPY) null mice showed decreased KiSS-1 mRNA levels at the hypothalamus, whereas exposure to NPY increased expression of KiSS-1 in hypothalamic N6 cells. In sum, our present data further characterize the functional relevance and putative key mediators (such as leptin and NPY) of the metabolic regulation of the hypothalamic KiSS-1 system in the mouse.     

Protein kinase C: a putative new target for the control of human medullary thyroid carcinoma cell proliferation in vitro

We investigate the role of protein kinase C (PKC) in the control of medullary thyroid carcinoma (MTC) cell proliferation by a PKC inhibitor, Enzastaurin, in human MTC primary cultures and in the TT cell line. We found that PKC inhibition reduces cell proliferation by inducing caspase-mediated apoptosis and blocks the stimulatory effect of IGF-I on calcitonin secretion. Enzastaurin reduces PKCβII (Thr500) phosphorylation, indicating a direct involvement of this isoform as well as the phosphorylated levels of Akt (Ser 473) and glycogen synthase kinase (Ser9), PKC pathway downstream targets and pharmacodynamic markers for PKC inhibition. PKCβII and PKCδ enzyme isoforms expression and localization were investigated. These data indicate that in vitro PKC is involved in the control of human MTC proliferation and survival by modulating apoptosis, with a mechanism that implicates PKCβII inhibition and translocation in different subcellular compartments. Targeting PKC may represent a useful therapeutic approach for controlling MTC proliferation.